Applied Biochemistry and Biotechnology (v.167, #3)

Designer Xylanosomes: Protein Nanostructures for Enhanced Xylan Hydrolysis by Shara D. McClendon; Zichao Mao; Hyun-Dong Shin; Kurt Wagschal; Rachel R. Chen (395-411).
This work reports the successful design, construction, and application of multi-functional, self-assembling protein complex, termed xylanosomes. Using the architecture of cellulosomes as template, these structures were designed specifically for hemicellulose hydrolysis. Four different xylanosomes were developed, with up to three different hemicellulase activities combined into a single structure. Each xylanosome was composed of two native or chimeric hemicellulases and tested on wheat arabinoxylan or destarched corn bran for enzymatic hydrolysis. After 24-h incubation, soluble sugars released from arabinoxylan increased up to 30 % with xylanosomes containing a xylanase and bi-functional arabinofuranosidase/xylosidase over the corresponding free, unstructured enzymes. Additionally, xylanosomes with a xylanase and a ferulic acid esterase removed between 15 and 20 % more ferulic acid from wheat arabinoxylan than free enzymes. Furthermore, xylanosomes exhibited synergy with cellulases on destarched corn bran, suggesting a possible use of these nanostructures in cellulose hydrolysis.
Keywords: Xylan hydrolysis; Cellulosome; Xylanosome; Hemicellulases; Lignocelluloses bioprocessing

Production of Potential Vaccine Against Dermatobia hominis for Cattle by Nelson L. M. Fernandes; Silvio M. Zanata; Milton Rönnau; Carlos R. Soccol; Ashok Pandey; Vanete Thomaz-Soccol (412-424).
The present study aimed to detect and characterize antigenic proteins and to assess their activity as preventive vaccines against dermatobiosis. Polyclonal antibodies were produced against three larval instars (L1, L2, L3), and their antigenic proteins were assessed for reactivity. Polyclonal antibodies produced in animals immunized with extracts were analyzed, and L3-derived antibodies showed proteins with better antigenic responses. The study of reactivity using immunodetection showed that the 50-kDa protein had the highest antigenicity. This protein was purified and subjected to mass spectrometry, and the sequences obtained were compared with those in the databases available. No similarities were found with existing sequences. Subsequently, large quantities of purified protein were used to immunize cattle. Vaccine effectiveness was evaluated by comparing the number of cutaneous nodules formed in the control group and immunized animals. The antigen produced proved a promising candidate for vaccine production, with 90.67 % efficacy. Immunohistochemistry of antigen–antibody reaction in larval sections showed epitopes all over larval tissues.
Keywords: Dermatobia hominis ; Antigen selection; Immunohistochemistry; Cattle; Vaccine

The global pool of intracellular metabolites is a reflection of all the metabolic functions of an organism. In the absence of in situ methods capable of directly measuring metabolite pools, intracellular metabolite measurements need to be performed after an extraction procedure. In this study, we evaluated the optimization of technologies for generation of a global metabolomics profile for intracellular metabolites in Klebsiella oxytoca. Intracellular metabolites of K. oxytoca were extracted at the early stationary phase using six different common extraction procedures, including cold methanol, boiling ethanol, methanol/chloroform combinations, hot water, potassium hydroxide, and perchloric acid. The metabolites were subsequently collected for further analysis, and intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. During analysis, the stability of metabolites extracted using cold methanol was clearly higher than that obtained by other extraction methods. For the majority of metabolites, extracts generated in this manner exhibited the greatest recovery, with high reproducibility. Therefore, the use of cold ethanol was the best extraction method for attaining a metabolic profile. However, in another parallel extraction method, perchloric acid may also be required to maximize the range of metabolites recovered, particularly to extract glucose 1-phosphate and NADPH.
Keywords: Klebsiella oxytoca ; Intracellular metabolites; LC/Q-TOF-MS; Metabolic profiling; Extraction

Unraveling the Rationale Behind Organic Solvent Stability of Lipases by Debamitra Chakravorty; Saravanan Parameswaran; Vikash Kumar Dubey; Sanjukta Patra (439-461).
Organic solvent-stable lipases have pronounced impact on industrial economy as they are involved in synthesis by esterification, interesterification, and transesterification. However, very few of such natural lipases have been isolated till date. A study of the recent past provided few pillars to rely on for this work. The three-dimensional structure, inclusive of the surface and active site, of 29 organic solvent-stable lipases was analyzed by subfamily classification and protein solvent molecular docking based on fast Fourier transform correlation approach. The observations revealed that organic solvent stability of lipases is their intrinsic property and unique with respect to each lipase. In this paper, factors like surface distribution of charged, hydrophobic, and neutral residues, interaction of solvents with catalytically immutable residues, and residues interacting with essential water molecules required for lipase activity, synergistically and by mutualism contribute to render a stable lipase organic solvent. The propensity of surface charge in relation to stability in organic solvents by establishing repulsive forces to exclude solvent molecules from interacting with the surface and prohibiting the same from gaining entry to the protein core, thus stabilizing the active conformation, is a new finding. It was also interesting to note that lipases having equivalent surface-exposed positive and negative residues were stable in a wide range of organic solvents, irrespective of their LogP values.
Keywords: Accessible surface area; Active site; Charge; Amino acid residues; Structure; Subfamily; Surface exposure

To increase the level of adsorption of cadmium ions to the surface of Escherichia coli, we fused cyanobacterial metallothioneins, SmtA (from Synechococcus elongatus PCC 3601) and MtnA (from Synechococcus vulcanus) to the E. coli cell surface using a Lpp′-OmpA-based display system. E. coli strains expressing Lpp′-OmpA–SmtA-linker-ChBD (chitin-binding domain from Bacillus pumillus SG2 chitinase S; chiS) and Lpp′-OmpA–MtnA-linker-ChBD on their surface adsorbed more cadmium compared to the E. coli cells expressing only the Lpp′-OmpA-linker-ChBD hybrid. These constructs also were bound to chitin through their chitin-binding domain, allowing them to be immobilized on a chitin matrix. We assessed surface presentation of Lpp′-OmpA–SmtA-linker-ChBD, Lpp′-OmpA–MtnA-linker-ChBD, and Lpp′-OmpA-linker-ChBD using immunostaining. The Lpp′-OmpA–SmtA-linker-ChBD chimera adsorbed metal and was bound to chitin with the highest efficiency compared to the other chimeras, suggesting that it is an effective bioadsorbent. This is the first example of coupling metal adsorption with cell immobilization using a whole-cell bioadsorbent.
Keywords: Cadmium; Surface display; Metallothionein; Synechococcus; Adsorption; Immobilization

Cell Culture Tracking by Multivariate Analysis of Raw LCMS Data by François-Thomas Michaud; Pierre Claver Havugimana; Carl Duchesne; François Sanschagrin; Alice Bernier; Roger C. Lévesque; Alain Garnier (474-488).
Liquid chromatography mass spectrometry (LCMS) is a powerful technique that could serve to rapidly characterize cell culture protein expression profile and be used as a process monitoring and control tool. However, this application is often hampered by both the sample proteome and the LCMS signal complexities as well as the variability of this signal. To alleviate this problem, culture samples are usually extensively fractionated and pretreated before being analyzed by top-end instruments. Such an approach precludes LCMS usage for routine on-line or at-line application. In this work, by applying multivariate analysis (MA) directly on raw LCMS signals, we were able to extract relevant information from cell culture samples that were simply lyzed. By using the recombinant adenovirus production process as a model, we were able to follow the accumulation of the three major proteins produced, identified their accumulation dynamics, and draw useful conclusions from these results. The combination of LCMS and MA provides a simple, rapid, and precise means to monitor cell culture.
Keywords: LCMS; Multivariate analysis; Protein production; Virus production; Cell culture monitoring

The stability of enzymes remains a critical issue in biotechnology. Compared with the strategies for obtaining stable enzymes, chemical modification is a simple and effective technique. In the present study, chemical modification of horseradish peroxidase (HRP) was carried out with pyromellitic anhydride. HRP has achieved a prominent position in the pharmaceutical, chemical, and biotechnological industries. In this study, the effect of chemical modification on thermal stability, structure, and function of the enzyme was studied by fluorescence, circular dichroism, and absorbance measurements. The results indicated a decrease in compactness of the structure and a considerable enhancement in thermal stability of HRP below 60 °C. It seems the charge replacement and introduction of the bulky group bring about the observed structural and the functional changes.
Keywords: Chemical modification; Horseradish peroxidase; Pyromellitic anhydride; Thermal stability

Construction of Human Nonimmune Library and Selection of scFvs Against IL-33 by Qing Yuan; Li Huang; Xu Wang; Yuchuan Wu; Yan Gao; Chengwen Li; Siji Nian (498-509).
Interleukin (IL) 33 plays very important roles in inflammatory and allergic diseases. To select human single-chain Fv fragments (scFvs) against IL-33, a nonimmune phage library system was constructed. The full-length cDNA library was synthesized for amplification of the variable heavy chain (VH) and variable light chain (VL). By overlapping extension PCR for splicing VH and VL, the full-length scFv library DNA were amplified and then transformed into Escherichia coli TG1. The scFv library was constructed successfully which contained 2.5 × 108 independent clones with full-length scFv inserts. The results of fingerprint maps of the scFvs by BstN I and DNA sequencing from the library at random proved that the library was diverse. The human IL-33 was amplified, expressed, and purified. The purified IL-33 with bioactivity was biotinylated and used as antigen for selection of scFv library by phage display. After three rounds of affinity selection, about 30 % of clones have specific binding activity with IL-33. Five of those with good binding activity were transformed into E. coli strain HB2151 for soluble expression. The selected scFvs were further identified by western blot and sequencing. Those selected scFvs could be used for further research of their effect on inflammatory and allergic diseases such as asthma by blockade of IL-33.
Keywords: ScFv; IL-33; Nonimmune library

To meet the increasing demands of sophorolipids as biosurfactants and bioactive compounds, it is necessary to obtain higher and more specific sophorolipid-producing strains. One sophorolipid-producing strain, Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 (Y2A), was mutated by low-energy nitrogen ion beam implantation. Eighteen mutants produced 20 % more sophorolipids than the wild strain, and one mutant, N3-18, produced the highest yield of sophorolipids, 104 g/l, in a shaking flask, which increased by 84.71 % than the wild strain, and further elevated to 135 g/l in a 5-l bioreactor. High performance liquid chromatography analysis showed that the composition of every sophorolipid mixture from different strains was similar, while the contents of most components from mutants were higher than that from the wild strain. Two mutants, N1-32 and N3-18, produced more acidic sophorolipid components; three lactonic sophorolipid molecules with good anticancer activities were greatly enhanced in several mutants, especially monoacetylated lactonic sophorolipid with a C18 monounsaturated fatty acid, which were enhanced by 153 and 211 % in strains N1-32 and N3-18. Low-energy nitrogen ion beam implantation was efficient for obtaining a variety of high and specific sophorolipid-producing mutants to be applied in food, cosmetic, environmental, and pharmaceutical sectors.
Keywords: Sophorolipids; Nitrogen ion beam implantation; Mutation breeding; Composition of sophorolipids

Enhanced Recovery and Purification of P(3HB-co-3HHx) from Recombinant Cupriavidus necator Using Alkaline Digestion Method by Siti Nor Syairah Anis; M. I. Nurhezreen; K. Sudesh; A. A. Amirul (524-535).
A simple, efficient and economical method for the recovery of P(3HB-co-3HHx) was developed using various chemicals and parameters. The initial content of P(3HB-co-3HHx) in bacterial cells was 50–60 wt%, whereas the monomer composition of 3HHx used in this experiments was 3–5 mol%. It was found that sodium hydroxide (NaOH) was the most effective chemical for the recovery of biodegradable polymer. High polyhydroxyalkanoate purity and recovery yield both in the range of 80–90 wt% were obtained when 10–30 mg/ml of cells were incubated in NaOH at the concentration of 0.1 M for 60–180 min at 30 °C and polished using 20 % (v/v) of ethanol.
Keywords: Polyhydroxyalkanoates; Cupriavidus ; Alkaline digestion; Recovery; 3HHx

Mushroom Polysaccharides and Lipids Synthesized in Liquid Agitated and Static Cultures. Part I: Screening Various Mushroom Species by Panagiota Diamantopoulou; Seraphim Papanikolaou; Maria Kapoti; Michael Komaitis; George Aggelis; Antonios Philippoussis (536-551).
The effect of four synthetic media containing glucose (initial concentration 30 g l−1) on mycelial growth, exopolysaccharides (EPS) and cellular lipids production was examined in 11 mushroom species after 12 and 16 days of culture in static- and shake-flasks. Fatty acid analysis of cellular lipids produced was also performed. Agitation had a positive effect on biomass production, glucose consumption and lipid biosynthesis. Media that favoured the production of biomass were not suitable for EPS biosynthesis and vice versa. Biomass values varied from ∼1.0 g l−1 (Lentinula edodes) to ∼19 g l−1 (Pleurotus ostreatus), while the highest EPS quantity achieved ranged between 1.6 and 1.8 g l−1 (for Ganoderma lucidum and L. edodes, respectively). Quantities of total cellular lipids varied between 2.5 and 18.5 % w/w, in dry mycelial mass for the fungi tested. Lipid in dry weight values were influenced by the medium composition. Cellular lipids presented noticeable quantities of poly-unsaturated fatty acids like linoleic acid. Compared to most of the mushrooms tested, lipids of Volvariella volvacea were more saturated. The ability of several mushroom species of our study to produce in notable quantities the above-mentioned added-value compounds renders these fungi worthy for further investigations.
Keywords: Ascomycetous; Basidiomycetous; Biomass; Cellular lipids; Exopolysaccharides; Submerged cultures

Purification of Papain Using Reactive Green 5 Attached Supermacroporous Monolithic Cryogel by Deniz Aktaş Uygun; Begüm Akduman; Murat Uygun; Sinan Akgöl; Adil Denizli (552-563).
Supermacroporous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] monolithic cryogel was prepared by radical cryocopolymerization of HEMA with N,N′-methylene bisacrylamide as crosslinker. Reactive Green 5 dye was immobilized to the cryogel with nucleophilic substitution reaction, and this dye attached cryogel column was used for affinity purification of papain from Carica papaya latex. Reactive Green 5-immobilized poly(HEMA) cryogel was characterized by swelling studies, Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray analysis. Maximum papain adsorption capacity was found to be 68.5 mg/g polymer while nonspecific papain adsorption onto plain cryogel was negligible (3.07 mg/g polymer). Papain from C. papaya was purified 42-fold in single step with dye attached cryogel, and purity of papain was shown by silver-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
Keywords: Dye ligand; Cryogel; Papain; Reactive green 5; Carica papaya

Methyl ethyl ketone (MEK) and methyl iso-butyl ketone (MIBK) constitute significant proportion of the total VOC emissions from manufacturing and application processes of surface coatings. Biodegradation of MEK and MIBK using an acclimatized mixed culture was evaluated, under aerobic condition. Biodegradation studies were carried out using MEK and MIBK as single substrates and in combination. Mixed-pollutant studies were conducted in MEK-dominated system, MIBK-dominated system, and MEK–MIBK equi-concentration systems to understand the concentration-dependent interaction of these compounds in a biosystem. Experimental data obtained from single-pollutant system was used to estimate the biokinetic parameters, viz. μ max, K s , K i , and Y T , for these compounds. Among the several bio-kinetic models tested, Monod inhibition model was best suited for predicting the biodegradation of these two VOCs. Four multiple-substrate models, viz. no-interaction, competitive, un-competitive, and non-competitive were used to study the nature of inhibition for different combinations of these compounds. The biodegradation of MEK and MIBK mixtures was found to be best described by competitive inhibition model. However, the predictions were not very good for systems where MEK concentration was higher than MIBK concentration.
Keywords: Bio-degradation; Inhibition; Rate kinetics; Acclimatized mixed culture; Methyl ethyl ketone; Methyl iso-butyl ketone

Schiff base ligand (H3L) was prepared from the condensation reaction of protochatechualdehyde (3,4-dihydroxybenzaldhyde)with 2-amino phenol. From the direct reaction of the ligand (H3L) with Co(II), Ni(II) and Cu(II) chlorides, and Fe(III)and Zn(II)nitrates in 2 M/1 L molar ratio, the five new neutral complexes were prepared. The characterization of the newly formed compounds was done by 1H NMR, UV–Vis, and IR spectroscopy and elemental analysis. The in vitro antibacterial activity of the metal complexes was studied and compared with that of free ligand.
Keywords: Schiff base complexes; Synthesis; Catechol; Spectra; Biological evaluation

Coenzyme Regeneration in Hexanol Oxidation Catalyzed by Alcohol Dehydrogenase by Ana Vrsalović Presečki; Katja Makovšek; Đurđa Vasić-Rački (595-611).
The enzymatic ways of coenzyme regeneration include the addition of a second enzyme to the system or the addition of the co-substrate. In the present study, both methods of enzymatic coenzyme (NAD+) regeneration were studied and compared in the reaction of hexanol oxidation catalyzed by alcohol dehydrogenase (ADH). As a source of ADH, commercial isolated enzyme and the whole baker’s yeast cells were used. First, coenzyme regeneration was employed in the reaction of acetaldehyde reduction catalyzed by the same enzyme that catalyzed the main reaction, and then NAD+ regeneration was applied in the reaction of pyruvate reduction catalyzed by l-lactate dehydrogenase (l-LDH). Hexanal was obtained as the product of hexanol oxidation catalyzed by isolated ADH while hexaonic acid was detected as a product of the same reaction catalyzed by baker’s yeast cells. All of the used biocatalysts were kinetically characterized. The mass reactions were described by the mathematical models. All models were validated in the batch reactor. One hundred percent hexanol conversion was obtained using permeabilized yeast cells using both methods of cofactor regeneration. By using isolated enzyme ADH, the higher conversion was achieved in a system with cofactor regeneration catalyzed by l-LDH.
Keywords: Yeast; Coenzyme regeneration; Enzyme kinetics; Alcohol dehydrogenase; Modeling; Alcohol oxidation

Manipulation of the Conformation and Enzymatic Properties of T1 Lipase by Site-Directed Mutagenesis of the Protein Core by Roswanira Abdul Wahab; Mahiran Basri; Raja Noor Zaliha Raja Abdul Rahman; Abu Bakar Salleh; Mohd Basyaruddin Abdul Rahman; Leow Thean Chor (612-620).
In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.
Keywords: Hydrophobic residue; Hydrophobic core; T1 lipase; Protein design; Conformational stability

Network-Based Identification of Novel Connections Among Apoptotic Signaling Pathways in Cancer by Nan Wang; Huai-long Xu; Xu Zhao; Xin Wen; Feng-tian Wang; Shu-ya Wang; Lei-lei Fu; Bo Liu; Jin-ku Bao (621-631).
MicroRNAs (miRNAs), highly conserved, non-coding endogenous RNA and nearly ~22 nucleotides (nt) in length, are well-known to regulate several apoptotic pathways in cancer. In this study, we computationally constructed the initial human apoptotic PPI network by several online databases, and further integrated these high-throughput datasets into a Naïve Bayesian model to predict protein functional connections. Based on the modified apoptotic network, we identified several apoptotic hub proteins such as TP53, SRC, M3K3/5/8, cyclin-dependent kinase2/6, TNFR16/19, and TGF-β receptor 1/2. Subsequently, we identified some microRNAs that could target the aforementioned apoptotic hub proteins by using TargetScan, PicTar, and Diana-MicroH. In conclusion, these results demonstrate the PPI network-based identification of new connections amongst apoptotic pathways in cancer, which may shed new light on the intricate relationships between core apoptotic pathways and some targeted miRNAs in human cancers.
Keywords: MicroRNA (miRNA); Cancer; Apoptosis; Protein–protein interaction (PPI) network; Naïve Bayesian model

Evaluation of Amino Acid O-Phosphoserine as Ligand for the Capture of Immunoglubulin G from Human Serum by Igor Tadeu Lazzarotto Bresolin; Sonia Maria Alves Bueno (632-644).
The amino acid ortho-phosphoserine (OPS) immobilized on agarose gel was evaluated as a ligand for adsorption of polyclonal human immunoglobulin G (IgG) from human serum in the presence of low ionic strength buffers. Screening of buffer systems showed sodium phosphate as the buffer that exhibited higher IgG purity values. Through breakthrough curve analysis for agarose-OPS (feeding of 31.93 mg of total protein per mL of gel), a purification factor of 5.4 with an IgG purity of 89 % was obtained (based on IgG, IgM, IgA, HSA, and Trf nephelometric analysis). IgG adsorption equilibrium studies showed that these data followed the Langmuir-Freundlich model, with cooperativity parameter (n) equal to 1.74, indicating the presence of positive cooperativity, probably due to multipoint interactions. The maximum IgG binding capacity was 24.2 mg mL−1, near the value for the bioaffinity ligand protein A. The agarose-OPS adsorbent provides an attractive alternative for capturing of IgG from human serum.
Keywords: O-phosphoserine; Amino acid; Human immunoglobulin G; Adsorption; Purification; Human serum

The release of colored wastewater represents a serious environmental problem and public health concern. Color removal from textile wastewater has become a big challenge over the last decades, and up to now, there is no single and economically attractive treatment method that can effectively decolorize the wastewater. Effluents from textile manufacturing, dyeing, and finishing processes contain high concentrations of biologically difficult-to-degrade or even inert auxiliaries, chemicals like acids, waxes, fats, salts, binders, thickeners, urea, surfactants, reducing agents, etc. The various chemicals such as biocides and stain repellents used for brightening, sequestering, anticreasing, sizing, softening, and wetting of the yarn or fabric are also present in wastewater. Therefore, the textile wastewater needs environmental friendly, effective treatment process. This paper provides a critical review on the current technology available for decolorization and degradation of textile wastewater and also suggests effective and economically attractive alternatives.
Keywords: Textile wastewater; Toxicity; Biodegradation; Treatment; Problems

Five laccase enzyme isoforms were isolated and purified to homogeneity from the cladodes of xerophytic Cereus pterogonus and Opuntia vulgaris plant species. Catalytic activity of all isoforms was enhanced 40 % by 1 mM Cu2+ and 1 mM Mn2+, whereas the activity was inhibited 100 % by 10 mM Fe2+. Enzyme was found stable in 4 M urea and exhibited inactivity of 50 % in 8 M urea concentration. Ethylenediaminetetraacetic acid and cysteine-HCl were able to completely inhibit the enzyme activity at 1 mM and 100 μM, respectively. Preheated enzyme samples showed enhanced and stable catalytic activity in the presence of divalent cations over a period of 30 min compared with controls. In the presence of metal ions (1 mM Cu2+ and 1 mM Mn2+), the preheated enzyme forms (60–90 °C) achieved 97 % of Malachite green and 98.75 % of Indigo blue (both at 2 %, w/v) dye decolorization in 12 h.
Keywords: Laccase; Malachite green; Indigo blue; Dye decolorization; Thermostable xerophyte